Development of Chromosome-specific Cytogenetic Markers and Merging of Linkage Fragments in Papaya

Development of Chromosome-specific Cytogenetic Markersand Merging of Linkage Fragments in Papaya

Ching Man Wai & Ray Ming & Paul H. Moore &

Robert E. Paull & Qingyi Yu

Received: 22 April 2010 /Accepted: 4 July 2010 /Published online: 31 July 2010# Springer Science+Business Media, LLC 2010

Abstract Carica papaya L. is a tropical and sub-tropicalfruit-tree crop with a small genome and nine pairs ofchromosomes. The transgenic cultivar ‘SunUp’ has beensequenced and three high-density genetic maps are avail-able for mapping agronomically and economically-important traits. However, the small size and similarmorphology of papaya chromosomes hinder their identifi-cation and few cytological resources are available forintegration of genetic and cytogenetic information. Fluo-rescence in situ hybridization (FISH) was performed onmitotic metaphase chromosomes using BAC clones harbor-ing mapped simple sequence repeat (SSR) markers asprobes. A total of 104 BAC clones covering all 12 linkagegroups (LGs) were tested and 12 of them, that gave a singlespecific signal, were chosen as representative of the 12 LGs

of the SSR genetic map. This set of chromosome-specificDNA markers acted as a foundation for papaya chromo-some karyotyping and re-assigning orientation of LGs.Chromosome-specific markers allowed us to assign theminor LGs 10, 11, and 12 to major LGs 8, 9, and 7,respectively. We thus reduced the number of LGs in thegenetic map to nine, corresponding to the haploid numberof papaya chromosomes. We also tested the relative orderof DNA markers on minor LGs 10 and 11 to place them ontop of LGs 8 and 9 in the correct orientation. RibosomalDNAs (rDNAs), a set of major cytogenetic markers, werepositioned on specific papaya chromosomes. The 25SrDNA showed strong signals at the constriction site of asingle pair of chromosomes identified as LG 2 by LG 2-specific BAC clone. The 5S rDNA showed strong signalson two pairs of chromosomes that are syntenic with LG 4-and LG 5-specific BAC clones. This integrated map willfacilitate genome assembly, quantitative trait locus (QTL)mapping, and the study of cytological, physical and geneticdistance relationships between papaya chromosomes.

Keywords Carica papaya .

Chromosome-specific markers . Cytogenetics .

Fluorescence in situ hybridization (FISH) . Genetic map

Introduction

Cytogenetics was used before the discovery of DNA structure(Watson and Crick 1953) to correlate visibly distinctchromosomal segments with a number of genetic traits inmaize (Creighton and McClintock 1931; McClintock 1931;McClintock and Hill 1931). Chromosome identificationallowed scientists to study recombination events and thespatial arrangement of each chromosome (Lysak et al. 2001)

Communicated by: Scott Jackson

Electronic supplementary material The online version of this article(doi:10.1007/s12042-010-9054-1) contains supplementary material,which is available to authorized users.

C. M. Wai : R. E. PaullDepartment of Tropical Plant and Soil Sciences,University of Hawaii,Honolulu, HI 96822, USA

C. M. Wai : R. Ming : P. H. Moore :Q. YuHawaii Agriculture Research Center,Waipahu, HI 96797, USA

R. MingDepartment of Plant Biology,University of Illinois at Urbana-Champaign,Urbana, IL 61801, USA

Q. Yu (*)Texas A&M University, AgriLife Research Center,Weslaco, TX 78596-8344, USAe-mail: [email protected]

Tropical Plant Biol. (2010) 3:171–181DOI 10.1007/s12042-010-9054-1

within and between species (Hizume et al. 2002; Lim et al.2000). Traditionally, chromosome banding techniques (e.g.C- and G-banding) and morphological characteristics (e.g.chromosome size, arm ratio) played important roles inchromosome identification.

Repetitive sequences such as microsatellites or simplesequence repeats (SSRs) and ribosomal DNA (rDNA), havebeen used as landmarks for chromosome karyotyping (Hanet al. 2008; Islam-Faridi et al. 2007; Ren et al. 2009). Dueto its conserved sequences in plant, rDNA has primarilybeen used with data on chromosome lengths and arm ratiosto characterize specific chromosomes (Han et al. 2008;Islam-Faridi et al. 2007; Wang et al. 2008). The tandemarray of 45S rDNA forms the nucleolus organizing region(NOR) associated with the nucleolus while 5S rDNA is acomponent of large subunit of ribosome for translation.Although rDNA sequences are conserved, the number andposition of rDNA sequences vary among species orcultivars (Chung et al. 2008; Koornneef et al. 2003; Rosatoet al. 2008). The cytogenetic location of ribosomal DNAcombined with geographical co-distribution, predicted theA genome ancestor of tetraploid Arachis specie (Robledo etal. 2009). This method also facilitated the study ofpolyploidization of glycine species at chromosomal level(Krishnan et al. 2001).

Chromosome identification in plants gives a broad viewof chromatin structure organization but detailed informationon gene ordering and recombination can only be providedby genetic maps. The DNA markers, such as amplifiedfragment length polymorphism (AFLP), single nucleotidepolymorphism (SNPs) and sequence-tagged microsatellites,are commonly used in genetic maps. These are importantresources for mapping quantitative trait loci (QTL) control-ling agronomic traits (Cooper et al. 2009; Jansen et al.2009) and they facilitate genome assembly (InternationalRice Genome Sequencing Project 2005; Ming et al. 2008;Paterson et al. 2009;). Integrated genetic and cytogeneticmaps have been produced in plant in an attempt to assignLGs to chromosomes and to elucidate the relationshipbetween different types of chromosome structures andrecombination rates for genetic maps (Iovene et al. 2008;Kim et al. 2005a; Peters et al. 2009; Wang et al. 2006).

Currently, there are two main approaches for integratinggenetic and cytogenetic maps: (1) DNA markers arephysically mapped to chromosome regions in deletion ortranslocation stocks; and (2) large-insert clones with DNAmarkers are assigned directly to chromosomes by FISH.The former method has been used mainly in the analysis ofplant species having a large genome size and establishedcytogenetic stocks such as barley (Kunzel et al. 2000),maize (Chao et al. 1996) and wheat (Dieguez et al. 2006;Hohmann et al. 1994; Milla and Gustafson 2001; Saintenacet al. 2009). Chromosome morphological differences in

deletion or translocation lines can be distinguished easily inthese species due to the large size of their chromosomes.Chromosome arms of maize can be distinguished by thepattern of C-banding, G-banding (Kim et al. 2009) andHKG-banding (de Carvalho and Saraiva 1997). Thesepatterns facilitate the linkage of phenotypic traits tochromosome segments.

However, plant species with small chromosomes, suchas Arabidopsis, papaya and potato (Kato et al. 2005),cannot be visually analyzed but depend on newer methodsbased on large-insert clone libraries (such as BAClibraries). Storey (1953) reported that papaya chromosomes(2n=18) are similar in size during metaphase and theyappear to be metacentric or submetacentric (C. M. Wai, R.Ming, P. H. Moore, R. E. Paull, Q. Yu, unpublished).Recently, high resolution papaya pachytene spreads showedthat the distal ends of chromosomes are mostly euchromatic(Ming et al. 2008; Zhang et al. 2008) with centromericheterochromatic blocks. The small size and chromosomemorphological uniformity make it difficult to identifyindividual papaya chromosomes based on banding pattern,arm ratio, or chromosome length. With the development ofFISH, chromosome identification became more versatileand accurate by using repetitive sequences and large-insertclones as probes (Jiang et al. 1995; Lengerova et al. 2004).FISH gives reliable and routine results regardless of thechromosome size and the quality of chromosome prepara-tions (Jiang and Gill 2006). For example, the 12 pairs ofSilene latifolia chromosomes were distinguished by simul-taneously hybridizing mitotic chromosomes with threerepetitive sequences and five BAC clones (Lengerova etal. 2004). A set of chromosome-specific BAC clones weredeveloped in potato and have been used for mapping thechromosomal position of a potato late blight resistance gene(Dong et al. 2000). Chromosome identification usingchromosome-specific cytogenetic DNA markers have beendemonstrated in rice (Cheng et al. 2001), cotton (Wang etal. 2007), sorghum (Kim et al. 2002), tomato (Tang et al.2009), Antirrhinum majus (Zhang et al. 2005), potato(Dong et al. 2000) and common bean (Pedrosa et al.2003). The cytogenetic markers used in these species havebeen mainly developed from BAC clones containingmicrosatellite or AFLP genetic markers.

Recently, a draft genome sequence of transgenic papaya‘SunUp’ cultivar was released (Ming et al. 2008). Genomicresources, such as BAC libraries (Ming et al. 2001), ESTresources and three high-density genetic maps (Blas et al.2009; Chen et al. 2007; Ma et al. 2004) are also available.A high-density sequence-tagged genetic map of papaya wasconstructed using 706 microsatellite (or simple sequencerepeat, SSR) markers and the morphological marker of fruitcolor (Chen et al. 2007). However, due to variation in therecombination rate along chromosomes, gaps remained that

172 Tropical Plant Biol. (2010) 3:171–181

complicated the assembling of genetic markers on specificchromosomes. In this SSR genetic map, nine major linkagegroups (LG 1–9), which were hypothesized to represent thenine pairs of papaya chromosomes, and three minor LGs(LG 10–12) were constructed with a total length of1069.9 cM. In attempts to increase the genome coverageof this genetic map, an additional 277 AFLP markers wereintegrated into the SSR genetic map but these additionswere still unable to bridge the gaps between the major andminor LGs (Blas et al. 2009). The resulting incompletegenetic map could confound positional cloning genes ofinterest and QTL mapping of important traits.

We hypothesize that chromosome-specific cytogeneticmarkers derived from the existing SSR genetic map couldbe used with FISH techniques to identify individualchromosomes. Since the cytogenetic markers includemarker sequences, they will facilitate the physical assign-ment of the genetic map to chromosomes. This set ofcytogenetic markers might also be a good method forassigning the three minor LGs of SSR genetic map to thenine major ones as it allows direct visualization of FISHsignals on chromosomes (Jiang et al. 1995). A completelyassembled genetic map will facilitate the papaya genomeassembly, enhance the capacity for positional cloning ofgenes with important agronomic value, contribute to thecomparative genomics between papaya and other Brassi-cales species and allow for the study of relationshipsbetween chromatin structure and recombination rate alongthe chromosomes.

Results

Development of Chromosome-specific CytogeneticMarkers

The sequence-tagged high density genetic map generatedfrom an AU9 × SunUp F2 population was used to identifymarkers that might be useful for tagging of individualchromosomes (Chen et al. 2007). A total of 104 micro-satellite markers were selected from this genetic map thatcovered all 12 LGs (Supplementary File 1). With assistancefrom the integrated map (Yu et al. 2009), the BAC clonesharboring these microsatellite markers were identified.Most microsatellite markers were chosen from non-clustering regions or at the distal ends of each LG to avoidany BAC clones containing highly abundant repetitivesequences, which were mostly confined to marker-clusteredproximal regions of the LGs. BAC DNA was isolated andhybridized on mitotic metaphase chromosomes with the aidof Cot-1 DNA to suppress background signals producedfrom repetitive sequences. Among the 104 BAC clonesselected, 16 of them are located at genetic positions with a

cluster of five or more markers; while the rest 88 BACclones are located at genetic positions with less than fivemarkers. A total of 54 BAC clones gave specific FISHsignals on only one pair of chromosomes. Five of them arelocated at genetic positions with a cluster of five or moremarkers; while the remaining 49 clones that gave unam-biguous signals are located at genetic positions with lessthan five markers (Table 1).

Only BAC clones giving reproducible and unambiguousFISH signals were chosen as chromosome-specific markers.Among the 54 BAC clones showing unambiguous FISHsignals, 12 were chosen as the most reliable representationsof LG-1 to LG-12 of SSR genetic map (Fig. 1). The geneticpositions of 12 LG-specific BAC clones are locatedprimarily near or at the distal ends of the genetic map(Table 2). Notable exceptions to this observation were withLG 8 and LG 9 where the BAC clones were located nearerthe middle of the LGs.

Nine BAC clones, corresponding to the nine major LGs,were simultaneously hybridized on mitotic chromosomes ofa single nucleus in an attempt to integrate the genetic mapLGs with the chromosomes. Each BAC clone signal wasobserved on a single pair of chromosomes, suggesting thenine major LGs represent nine pairs of chromosomes(Fig. 1a) and that each of three minor LGs may be linkedto different major LGs. All signals observed were near or atthe distal ends of chromosomes.

Integration of Linkage Groups and Chromosomes

Once papaya chromosome-specific cytological markerswere established, the 12 LGs of the SSR genetic map wereassigned to nine pairs of papaya chromosomes. BAC clonesfrom the three minor LGs (10, 11 and 12) were screenedwith LG-specific cytogenetic markers from each of the ninemajor LGs (1 through 9) by FISH. This allowed determin-ing colocalization of LG signals on the same mitoticmetaphase chromosome.

By using this strategy, BAC clone 99D21 containingmicrosatellite marker CPM2098 from LG 10 and BACclone 35I09 containing microsatellite marker CPM766from LG 8 were observed as located at two opposite endsof a single pair of chromosomes. Thus, LG 10 wasrecognized as the north arm of LG 8 (Fig. 2a). Theorientation of LG10 on the top of LG8 was analyzed bysimultaneously hybridizing two BAC clones, 25C09 and99D21, from LG 10 and BAC clone 51E11 from the northarm of LG 8 to a meiotic pachytene chromosomepreparation (Fig. 2b). LG10 BAC clone 25C09 containingmicrosatellite marker P3K7484 (27 cM) was observed atthe chromosome end and BAC clone 99D21 carryingmarker CPM2098 (0 cM) located nearer to the LG 8 BAC51E11 than did BAC clone 25C09. This observation

Tropical Plant Biol. (2010) 3:171–181 173

indicated that LG 10 was linked to the top of of LG 8 in aninverted direction (Fig. 2c).

Minor LG 11wasmerged with another linkage group usingthe same method used for assigning LG 10 to a chromosome.BAC clone 39P03 from LG 11 and BAC clone 67F13 fromLG 9 were visualized at one end of a single pair of mitoticmetaphase chromosomes (Fig. 2d). The relative order ofmicrosatellite markers was resolved on meiotic pachytenechromosomes (Fig. 2e). Specifically, LG 11 BAC clone39P03 harboring microsatellite marker CPM1125 (27 cM)and BAC clone 09J08 containing marker P3K149 (0 cM)were pseudo-colored in red and green, respectively. BACclone 39P03 in green color was close to the telomeric regionof a pachytene chromosome while BAC clone 87B15containing microsatellite marker P3K4426 (6 cM) of LG 9in green color, was observed adjacent to LG 11 BAC clone09J08. Thus, the inverted minor LG 11 was placed on thetop of LG9 (Fig. 2f).

For the smallest LG, LG 12, FISH on a preparation ofmitotic metaphase chromosomes showed that LG 12 BACclone 1P02 and LG 7 BAC clone 24J15 were located atopposite ends of a single pair of chromosomes. Thisobservation suggested that LG 12 is located at the northarm of LG 7 (Fig. 3). Due to the small physical distancebetween the two ends of LG 12 and the limited number ofchromosome-specific BAC clones available for LG 12, theorientation of LG 12 on the north arm of LG 7 could not beverified with additional markers.

Localization of Ribosomal DNAs on Genetic Map

Arabidopsis thaliana rDNA sequences were used as aquery in a nucleotide BLAST search against the papayagenome sequence database to obtain papaya rDNA sequen-ces. Eight hits with an expectation value lower than e−10

were obtained for the 25S rDNA. A partial papaya 25SrDNA sequence was cloned and used for assigningnucleolus organizing region (NOR) on papaya chromo-somes. Only one pair of chromosomes was labeled and thesignals were located at constriction sites and close to the

middle of the chromosomes (Fig. 4a). The 25S rDNAsignals were syntenic to LG 2 BAC clone 15O14,suggesting that the NOR located at the center of LG 2.

More than 100 BLAST hits were obtained usingArabidopsis thaliana partial 5S rDNA sequence as thequery. The hit with the highest expectation values (super-contig 1275) contained more than 50 tandem repeats of thequery sequence. A partial 5S rDNA sequence was clonedfrom papaya genomic DNA and the plasmid was used as aprobe for FISH. Four strong signals were observed atinterstitial positions of two pairs of chromosomes (Fig. 4b).The 5S rDNA signals were syntenic to LG 4 and 5. On LG4, the 5S rDNA locus was flanked by BAC clonescontaining microsatellite markers P3K398 (31 cM) andCPM1024 (48 cM) (Fig. 4b). On LG 5, the 5S rDNA locuswas located between markers CPM988 (44 cM) andP6K911 (63 cM) (Fig. 4c).

Discussion

A saturated genetic map is essential for assembling thegenome sequences into pseudomolecules. Such a mapshould have the identical number of LGs with the numberof chromosomes. The papaya high density genetic mapcontains 706 SSR markers at a density of 1.9 markers perMb (Chen et al. 2007). However there are nine major andthree minor LGs for the nine pairs of chromosomes inpapaya, and adding 277 AFLP markers could not close anyof the three gaps (Blas et al. 2009). The inability to bridgethe gaps by linkage mapping might be due to highlyskewed marker segregation on those minor LGs, aninsufficient number of markers, or a large physical distancebetween gaps. Using BAC clones containing mappedmarkers and FISH techniques, the three minor LGs 10,11, and 12 were assigned to LGs 8, 9, and 7, respectively,consolidating the total number of LGs to nine. These newversion of the genetic map will help to finish sequencingthe papaya genome in foreseeable future as sequencing costcontinues to decline. It is a valuable resource for gene

Table 1 Relationship between clustering of microsatellite markers and specificity on FISH

No. of marker Specificity on FISH

a b c

Marker clustered with <5 markers at same genetic position 88 49 14 25

Marker clustered with ≥5 markers at same genetic position 16 5 5 6

Total 104 54 19 31

a Specific single signals on one pair of chromosomesbMajor signal on one pair of chromosomes with weak background on other chromosomesc Strong non-specific signal on more than one pair of chromosomes

174 Tropical Plant Biol. (2010) 3:171–181

Fig. 1 Papaya chromosomes labeled with linkage group-specificBAC clones. (a) Papaya chromosomes simultaneously hybridized withnine BAC clones that represent the major LGs 1–9. BAC clones15O14, 23B18, and 78D03 specific for LGs 2, 6 and 8, respectively,were labeled with biotin (red). BAC clones 96C17, 7H21, 43N18,57E17, 12M21, and 39C20 specific for LGs 1, 3, 4, 5, 7 and 9 werelabeled with DIG (green). Each LG-specific BAC clone showed signalon a single pair of chromosomes. (b–m) Papaya chromosomeshybridized with single LG-specific BAC clones. (b) Papaya chromo-somes hybridized with LG 1-specific BAC clone 96C17; (c) Papayachromosomes hybridized with LG 2-specific BAC clone 15O14; (d)Papaya chromosomes hybridized with LG 3-specific BAC clone

7H21; (e) Papaya chromosome hybridized with LG 4-specific BACclone 43N18; (f) Papaya chromosomes hybridized with LG 5-specificBAC clone 57E17; (g) Papaya chromosomes hybridize with LG 6-specific BAC clone 23B18; (h) Papaya chromosomes hybridized withLG 7-specific BAC clone 12M21; (i) Papaya chromosomes hybridizedwith LG 8-specific BAC clone 78D03; (j) Papaya chromosomeshybridized with LG 9-specific BAC clone 39C20; (k) Papayachromosomes hybridized with LG 10-specific BAC clone 99D21; (l)Papaya chromosomes hybridized with LG 11-specific BAC clone39P03; and (m) Papaya chromosomes hybridized with LG 12-specificBAC clone 1P02. Bar=2 μm

Tropical Plant Biol. (2010) 3:171–181 175

tagging and QTL mapping of agronomic traits in genomewide scale.

Meiotic pachytene chromosomes provide considerablehigher power of resolution for differentiating among

chromosomes than do mitotic metaphase chromosomes. InArabidopsis, metaphase chromosomes can resolve probesthat are 3 Mb apart while pachytene chromosomes canresolve probes that are only 75 kb apart (Schubert et al.

Fig. 2 Assignment of minor LGs to major LGs on mitotic and meioticchromosomes. (a) LG 8 BAC clone 35I09 (red) and LG 10 BACclone 99D21 (green) showed signals on one pair of metaphasechromosome by FISH. (b) The order of LG 8 BAC clone 51E11harboring microsatellite marker CPM1951 (green) and two LG 10BAC clones, 25C09 (P3K7484, green) and 99D21 (CPM2098, red)were revealed on pachytene chromosome. (c) The genetic andcytological positions of LG 8 marker CPM1951 (green) and two LG10 markers P3K7484 (green) and CPM2098 (red) are presented

diagrammatically (not to scale). (d) LG 9 BAC clone 67F13 (red) andLG 11 BAC clone 39P03 (green) are localized at one end of a singlepair of metaphase chromosomes. (e) The order of LG 9 BAC clone87B15 (P3K4426, green) and two LG 11 BAC clones, 39P03(CPM1125, green) and 09J08 (P3K149, red) are shown on pachytenechromosome. (f) The corresponding genetic and cytological positionsof LG 9 marker P3K4426 (green) and two LG 11 markers CPM1125(green) and P3K149 (red) were indicated on diagrammatically (not toscale). Bar=2 μm

Table 2 List of papaya chromosome-specific BAC clones

Linkagegroup

Total length of linkage group(cM)

Selected microsatellitemarker

Genetic positiona

(cM)BAC clone containing microsatellitemarker

1 145.0 CPM1737 112.6 96C17

2 138.8 CPM976 129.6 15O14

3 132.4 CPM746 0 7H21

4 120.6 CPM1111 91.0 43N18

5 103.6 CPM1657 20.7 57E17

6 100.2 CPM1010 3.4 23B18

7 96.4 CPM1846 27.9 12M21

8 91.8 CPM1564 46.9 78D03

9 64.5 CPM584/1129 31.2 39C20

10 27.1 CPM2098 0 99D21

11 26.8 CPM1125 26.6 39P03

12 21.4 P3K3510 0 1P02

a The genetic position of microsatellite marker on corresponding linkage group according to SSR genetic map (Chen et al. 2007)

176 Tropical Plant Biol. (2010) 3:171–181

2001). In maize chromosome 9, the resolution of metaphasechromosomes varied from 3.3 to 8.2 Mb while theresolution of pachytene chromosomes was at least275 kbp, which is 12 times greater than the resolution withmetaphase chromosomes (Danilova and Birchler 2008).Due to their higher resolving power, pachytene chromo-some preparations have been used in FISH to resolvemarker order on genetic maps (Islam-Faridi et al. 2002;Kim et al. 2005b), to construct cytogenetic maps (Koo et al.2008; Peters et al. 2009) and to study chromosomalrearrangements (Iovene et al. 2008; Tang et al. 2008;Walling et al. 2006). In the present study, the relative ordersof DNA markers of LG 9 with LG 11, and LG 8 with LG10 were observed on extended meiotic pachytene chromo-somes. These observations enabled us to place the minor

LGs on the top of major LGs in the well defined orientationthat could not be resolved on mitotic metaphase chromo-somes. All three minor LGs were located at the distal endsof assigned major LGs.

In our pachytene FISH results, no highly DAPI stainedregion was observed at the gap between LG 9 and LG 11and the gap between LG 8 and LG 10. In addition, we hadthe genomic sequence of the gap between LG 8 and 10 andused it to analyze the gene density and repeat content byRepeatMasker (Smit AFA, Hubley R and Green P, http://www.repeatmasker.org). The repetitive sequence density andgene content at the gap and its surrounding regions weresimilar (Wai CM, unpublished results). Thus, it is unlikelythat inability of map across the gaps was due to high repeatcontent or paucity of genes. In the artichoke female geneticmap, 20 LGs were constructed for 17 pairs of chromosomes.The authors of this paper suggested that the unassigned LGsmay due to the lack of polymorphism between parents in thegap regions or it might be due to overly stringent parametersused in genetic map construction (Portis et al. 2009). Thesehypotheses might also explain the appearance of minor LGsin the several papaya genetic maps.

This is the first report of using selected papaya BACclones for integrating mapped LGs to individual chromo-somes. In the present study, the use of chromosome-specificmarkers for FISH-based chromosome identification ishighly reproducible and does not require any specificstages of chromosome preparation as is required for bandstaining and arm ratio measurements. This approach can beused at different stages of mitosis or meiosis. In addition,arm measurements so useful with large chromosome plantsare unreliable for small chromosomes since chromosomelengths can vary so greatly to confound their nomenclature

Fig. 4 Positioning of ribosomal DNAs on genetic map by FISH.Ribosomal DNAs were hybridized with chromosome-specific BACclones simultaneously on mitotic metaphase chromosomes to revealtheir locations. (a) A single strong signal of 25S rDNA was observednear the middle of LG 2 by simultaneously hybridizing a plasmidcarrying partial 25S rDNA (green) and a LG 2-specific BAC clone15O14 (red). (b) A partial 5S rDNA sequence clone (red) showed fourmajor signals on two pairs of chromosomes. Two LG 4 BAC clones(green), 67N22 and 29G19, hybridized with 5S rDNA (red) onmetaphase chromosomes simultaneously. BAC clone 29G19 harbor-

ing microsatellite marker P3K398 (31 cM) showed specific signalswhile four signals were observed from BAC clone 67N22, whichcontains microsatellite marker CPM1024 (48 cM). The 5S rDNAsignal was flanked by these two LG 4 BAC clones. (c) The papayametaphase chromosomes were hybridized simultaneously with the 5SrDNA clone (red) and two LG 5-specific BAC clones (green), 60F11and 17E10. The 5S rDNA signals were located between the two LG 5-specific BAC clones, which carried microsatellite markers CPM988(44 cM) and P6K911 (63 cM). Bar=2 μm

Fig. 3 Physical assignment of LG 7 to LG 12 by FISH. LG 7 BACclone 24J15 (red) and LG 12 BAC clone 1P02 (green) were located atopposite ends of a single pair of chromosomes. Bar=2 μm

Tropical Plant Biol. (2010) 3:171–181 177

(Kim et al. 2009). Thus, the use of chromosome-specificmarkers in FISH is the most certain method for papayachromosome identification.

Papaya sequence-tagged genetic map has been producedfrom microsatellite markers developed from whole genomeshotgun sequences, BAC end sequences and subclones ofBAC clones. It was thus straight forward to anchor DNAmarkers to their corresponding BAC clones. The individualLGs of SSR genetic map could be distinguished on mitoticchromosomes by using only 12 LG-specific BAC clones asprobes. As suggested in Chen et al. (2007), our FISH resultconfirmed that the nine longest LGs represent the nine pairsof papaya chromosomes.

Microsatellite markers at various regions of papaya SSRgenetic map were chosen and tested for their ability toidentify specific papaya chromosomes. Previous studiesshowed that the distal ends of chromosomes are mainlyDAPI light-stained euchromatic regions containing low-repeat DNA (Ming et al. 2008; Zhang et al. 2008). Suchregions might be expected to show highly specific signalson chromosomes in FISH. This hypothetical chromosomestructure is consistent with our results showing that 24 of54 chromosome-specific cytogenetic markers were locatedat distal ends, where fewer microsatellite markers wereclustered. The phenomenon of chromosome specificmarkers near the chromosome ends has also been observedin rice (Cheng et al. 2001), sorghum (Kim et al. 2005a) andAntirrhinum majus (Zhang et al. 2005). In sorghum, among22 markers chosen at the distal end of LGs, 19 showedspecific FISH signals and only three showed signals withmoderate background without Cot-1 suppression. Hetero-chromatic regions have low recombination rate and thuslead to clustering of genetic markers. Heterochromaticregions also contain large amounts of repetitive sequencesthat produce non-specific signals when using FISH (Peterset al. 2009; Wang et al. 2006). Surprisingly, thechromosome-specific markers of LG 8 and LG 9, whichlocated in a marker clustering region, also gave unambig-uous signals on FISH. This result might be due to smallamount of repeat sequences (Lai et al. 2006; Nagarajan etal. 2008), or low percentage of heterochromatin area inthese regions (Ming et al. 2008). This may also explainwhy we obtained a higher number of LG-specific signalsthan expected from 31% of microsatellite markers chosen atgenetic positions having clusters of five or more markers.

In this present study, only one NOR site was observed inpapaya and is similar to the other plants in which one totwo NOR sites were reported. For example, one NOR sitewas reported in soybean (Griffor et al. 1991) and two NORsites were observed in A. thaliana (Koornneef et al. 2003)and barley (Taketa et al. 1999). In the BLAST search usingArabidopsis thaliana 25S rDNA as query against papayagenome sequence, only five hits were obtained with an

expectation value less than e−10. This suggested that thecopy number of 25S rDNA in papaya genome is either lowor may not be assembled into scaffolds. For papaya 5SrDNA in this study, two major sites were observed onmetaphase chromosomes while 13 sites were observed onhigh-resolution pachytene chromosome (Zhang et al. sameissue). Our BLAST search showed that more than 100scaffolds contained multiple copies of 5S rDNA sequence.This data supported the possibility of high number of rDNAsites in the papaya genome. The difference in number of 5SrDNA sites observed is likely due to the compaction ofchromosome structure at metaphase which leads to de-creased resolution. In addition, the two very strong signalsobserved on LG 4 and 5 may very well mask the weaksignals from other sites.

Since ribosomal DNA consists of highly repetitivesequences, it creates difficulties in genome assembly. Theability to identify the exact position of ribosomal DNA inpapaya genome enabled us to design different sequencingstrategy for those regions to fill the gap. Furthermore, it hasbeen reported that ribosomal DNA number and position canbe different among varieties or closely related species (deMelo and Guerra 2003; Rosato et al. 2008; Shishido et al.2000). Thus, the NOR and 5S rDNA may be goodlandmarks for karyotyping and studying the evolutionaryrelationship of chromosome structure between papaya andits related species.

The distribution of microsatellite markers in wheat,barley and rye were showed to be specific and correlatedwith certain chromosomal structures (heterochromatin,euchromatin and centromeres) (Cuadrado et al. 2008). Withthe aid of chromosome-specific cytogenetic markers, wewere able to locate ribosomal DNAs on the genetic mapand we will be able to extend this method to study thedistribution of other tandem repeat sequences, such asmicrosatellites and knob-associated repeats, on each chro-mosome without the complete genome sequence. Thisfinding will enhance the knowledge of papaya genomeorganization at the chromosomal level.

Methods

Plant Materials

The papaya cultivar ‘SunUp’ was germinated and planted atthe Hawaii Agriculture Research Center Kunia Substation(Kunia, HI, USA). Mature trees were grown in the field andhermaphrodite flower buds that were 6–8 mm in lengthwere collected for pachytene chromosome preparation.Shoot apical meristems of 6-8-week old seedlings grownin greenhouse were collected for mitotic metaphasechromosomes preparation.

178 Tropical Plant Biol. (2010) 3:171–181

Chromosome Preparation

Immature flowers (6–8 mm in length) were collected fromhermaphrodite trees and fixed in 3:1 (100% ethanol: glacialacetic acid) Carnoy’s solution. Microsporocytes at thepachytene stage were squashed in 60% acetic acid onFisherbrand Superfrost Plus® slides (Fisher scientific, PA).Glass coverslips were removed with a razor blade afterimmersing slides in liquid nitrogen. The chromosome slideswere stored at 4°C until hybridization and analysis.

Mitotic metaphase chromosomes were prepared fromshoot apical meristems (SAMs) of 6–8-week old plants.SAMs were treated with saturated 1,4-dichlorobenzene and1-bromonaphthalene for 2–3 h to accumulate prometaphaseand metaphase cells, and then fixed in Carnoy’s solution.Samples were then squashed on slides in 60% acetic acidand air-dried. All slides were stored at 4° until used.

Probe Selection and Labeling

Microsatellite marker-anchored BAC clones of each linkagegroup were chosen for testing signal specificity in FISHstudies. Five to 16markers were tested on the nine major LGs,while one to three markers were tested on the three minorLGs. All BAC clones used in FISH analysis were screenedfrom the ‘SunUp’ hermaphrodite BAC library (Ming et al.2001) and the microsatellite markers contained in the BACclones were selected based on the papaya SSR genetic map(Chen et al. 2007). BAC DNAwas isolated using the Qiagenplasmid midi kit (QIAGEN, CA) according to the manu-facturer’s instructions, and then labeled with either digox-igenin (DIG)-11-dUTP, or biotin-16-dUTP by DIG-, orBiotin-nick translation mix (Roche Diagnostics, IN).

Molecular Cloning of Ribosomal DNAs

Ribosomal DNA sequences in the papaya genome weresearched by using Arabidopsis thaliana 45S rDNA sequences(Accession: X52320) as query. Primers were designed onputative rDNA in the papaya supercontig 0 sequence(2,423,715-2,424,400) by Primer 3 program (Rozen andSkaletsky 2000) for amplifying partial 25S rDNA sequence.The sequence of the forward primer was 5′ ctc ccc att cga cccgtc 3′ and the reverse primer was 5′ gcc atc att ttc ggg g 3′.The PCR reactions consisting of 1X Taq polymerase Buffer,1 unit EconoTaq® polymerase (Lucigen, WI), 10 ng ‘SunUp’genomic DNA, 0.2 mM dNTPs, 0.2 μM forward primer andreverse primer were denatured at 95°C for 5 min, a 30 cycleof 95°C for 30 s, 55°C for 30 s and 72°C for 1 min, followedby 72°C for 7 min and stored at 4°C. The expected size bandof 685 bp was extracted from agarose gel by Illustra GFX™PCR DNA and gel band purification kit (GE Healthcare, PA)before being labeled by nick translation.

Papaya genome sequence was searched using Arabidopsisthaliana partial 5S rDNA sequence (Accession: AY130728.1)as query. The 5S rDNA sequence was amplified by specificprimer (5′ aac ctg cgg acc ata att taa 3′ and 5′ gta gta cta ggatgg gtg ac 3′) with same reaction conditions used for 25SrDNA. The expected band size of 140 bp was cloned intopGEM-T® vector (Invitrogen, CA) by E. coli (BL21)transformation. Transformed plasmid was amplified inE. coli and 1 μg of DNA was used for probe labeling.

Fluorescence in Situ Hybridization (FISH)

Mitotic and meiotic chromosome slides were pretreatedwith RNase (100 μg/mL) at 37°C for 1 h, followed bypepsin (5 μg/mL) at 37°C for 15 min and 4% paraformal-dehyde at room temperature for 10 min. After washingthree times at 5 min interval with 2X SSC, slides weredenatured in 70% deionized formamide at 80°C for 2 minand immediately dehydrated in an cold ethanol series (70%,90% and 95%) for 2 min each. Hybridization mixturesconsisted of 50% formamide, 2X SSC, 10% dextran sulfate,400 ng papaya C0t-1 DNA, 100–250 ng DIG-11-dUTP and/or biotin-16-dUTP labeled BAC DNAs. The DNA prepa-ration was denatured at 80°C for 5 min before applying it tochromosome preparations. The slides were incubated inOmniSlide thermal cycler (Thermo Electron Co., MA) at37°C for 18 h. DIG- and biotin-labeled BAC clones weredetected using 10 ng/mL anti-DIG-fluorescein antibody(Roche Diagnostics, IN) and streptavidin Alexa Fluor®568-conjugated antibody (Invitrogen, SD), respectively.Chromosomal DNA was counterstained with 4, 6-diamidino-2-phenylindole (1 μg/mL) in antifade Vector-shield solution (Vector Laboratories) and covered with aglass coverslip.

Cytological Analysis

Images were captured by 2.0 megapixels monochromeQuantiFire® camera (Optronics) attached to an OlympusBX51 epifluorescence microscope. Grey-scale images oneach color channel were captured, pseudo-colored andmerged by PictureFrame™ 2.2 image system (Optronics).

Acknowledgement This project was partially supported by a USDAT-STAR grant from the USDA-CSREES, Award # 2008-34135-19371, to the University of Hawaii at Manoa and a sub-award to theHawaii Agriculture Research Center.

References

Blas AL, Yu Q, Chen C, Veatch O, Moore PH, Paull RE, Ming R(2009) Enrichment of a papaya high-density genetic map withAFLP markers. Genome 52:716–725

Tropical Plant Biol. (2010) 3:171–181 179

Chao S, Gardiner JM, Melia-Hancock S, Coe EH Jr (1996) Physicaland genetic mapping of chromosome 9S in maize usingmutations with terminal deficiencies. Genetics 143:1785–1794

Chen C, Yu Q, Hou S, Li Y, Eustice M, Skelton RL, Veatch O, HerdesRE, Diebold L, Saw J, Feng Y, Qian W, Bynum L, Wang L,Moore PH, Paull RE, Alam M, Ming R (2007) Construction of asequence-tagged high-density genetic map of papaya for com-parative structural and evolutionary genomics in brassicales.Genetics 177:2481–2491

Cheng Z, Buell CR, Wing RA, Gu M, Jiang J (2001) Toward acytological characterization of the rice genome. Genome Res11:2133–2141

Chung MC, Lee YI, Cheng YY, Chou YJ, Lu CF (2008) Chromo-somal polymorphism of ribosomal genes in the genus Oryza.Theor Appl Genet 116:745–753

Cooper M, van Eeuwijk FA, Hammer GL, Podlich DW, Messina C(2009) Modeling QTL for complex traits: detection and contextfor plant breeding. Curr Opin Plant Biol 12:231–240

Creighton HB, McClintock B (1931) A correlation of cytological andgenetical crossing-over in Zea Mays. Proc Natl Acad Sci USA17:492–497

Cuadrado A, CardosoM, Jouve N (2008) Physical organisation of simplesequence repeats (SSRs) in Triticeae: structural, functional andevolutionary implications. Cytogenet Genome Res 120:210–219

Danilova TV, Birchler JA (2008) Integrated cytogenetic map ofmitotic metaphase chromosome 9 of maize: resolution, sensitiv-ity, and banding paint development. Chromosoma 117:345–356

de Carvalho CR, Saraiva LS (1997) High-resolution HKG-banding inmaize mitotic chromosomes. J Plant Res 110:417–420

de Melo NF, Guerra M (2003) Variability of the 5S and 45S rDNAsites in Passiflora L. species with distinct base chromosomenumbers. Ann Bot 92:309–316

Dieguez MJ, Altieri E, Ingala LR, Perera E, Sacco F, Naranjo T (2006)Physical and genetic mapping of amplified fragment length poly-morphisms and the leaf rust resistance Lr3 gene on chromosome6BL of wheat. Theor Appl Genet 112:251–257

Dong F, Song J, Naess SK, Helgeson JP, Gebhardt C, Jiang J (2000)Development and applications of a set of chromosome-specificcytogenetic DNA markers in potato. Theor Appl Genet101:1001–1007

Griffor MC, Vodkin LO, Singh RJ, Hymowitz T (1991) Fluorescent insitu hybridization to soybean metaphase chromosomes. PlantMol Biol 17:101–109

Han YH, Zhang ZH, Liu JH, Lu JY, Huang SW, Jin WW (2008)Distribution of the tandem repeat sequences and karyotyping incucumber (Cucumis sativus L.) by fluorescence in situ hybrid-ization. Cytogenet Genome Res 122:80–88

Hizume M, Shibata F, Matsusaki Y, Garajova Z (2002) Chromosomeidentification and comparative karyotypic analyses of four Pinusspecies. Theor Appl Genet 105:491–497

Hohmann U, Endo TR, Gill KS, Gill BS (1994) Comparison ofgenetic and physical maps of group 7 chromosomes fromTriticum aestivum L. Mol Genet Genomics 245:644–653

International Rice Genome Sequencing Project (2005) The map-basedsequence of the rice genome. Nature 436:793–800

Iovene M, Wielgus SM, Simon PW, Buell CR, Jiang J (2008) Chromatinstructure and physical mapping of chromosome 6 of potato andcomparative analyses with tomato. Genetics 180:1307–1317

Islam-Faridi MN, Childs KL, Klein PE, Hodnett G, Menz MA, KleinRR, Rooney WL, Mullet JE, Stelly DM, Price HJ (2002) Amolecular cytogenetic map of sorghum chromosome 1. Fluores-cence in situ hybridization analysis with mapped bacterialartificial chromosomes. Genetics 161:345–353

Islam-Faridi MN, Nelson CD, Kubisiak TL (2007) Referencekaryotype and cytomolecular map for loblolly pine (Pinus taedaL.). Genome 50:241–251

Jansen RC, Tesson BM, Fu J, Yang Y, McIntyre LM (2009) Defininggene and QTL networks. Curr Opin Plant Biol 12:241–246

Jiang J, Gill BS (2006) Current status and the future of fluorescence insitu hybridization (FISH) in plant genome research. Genome49:1057–1068

Jiang J, Gill BS, Wang GL, Ronald PC, Ward DC (1995) Metaphaseand interphase fluorescence in situ hybridization mapping of therice genome with bacterial artificial chromosomes. Proc NatlAcad Sci USA 92:4487–4491

Kato A, Vega JM, Han F, Lamb JC, Birchler JA (2005) Advances inplant chromosome identification and cytogenetic techniques.Curr Opin Plant Biol 8:148–154

Kim JS, Childs KL, Islam-Faridi MN, Menz MA, Klein RR, Klein PE,Price HJ, Mullet JE, Stelly DM (2002) Integrated karyotyping ofsorghum by in situ hybridization of landed BACs. Genome45:402–412

Kim JS, Islam-Faridi MN, Klein PE, Stelly DM, Price HJ, Klein RR,Mullet JE (2005a) Comprehensive molecular cytogenetic analy-sis of sorghum genome architecture: distribution of euchromatin,heterochromatin, genes and recombination in comparison to rice.Genetics 171:1963–1976

Kim JS, Klein PE, Klein RR, Price HJ, Mullet JE, Stelly DM (2005b)Molecular cytogenetic maps of sorghum linkage groups 2 and 8.Genetics 169:955–965

Kim H, Choi SR, Bae J, Hong CP, Lee SY, Hossain MJ, Van NguyenD, Jin M, Park BS, Bang JW, Bancroft I, Lim YP (2009)Sequenced BAC anchored reference genetic map that reconcilesthe ten individual chromosomes of Brassica rapa. BMCGenomics 10:432

Koo DH, Jo SH, Bang JW, Park HM, Lee S, Choi D (2008)Integration of cytogenetic and genetic linkage maps unveils thephysical architecture of tomato chromosome 2. Genetics179:1211–1220

Koornneef M, Fransz P, de Jong H (2003) Cytogenetic tools forArabidopsis thaliana. Chromosome Res 11:183–194

Krishnan P, Sapra VT, Soliman KM, Zipf A (2001) FISH mapping ofthe 5S and 18S-28S rDNA loci in different species of Glycine.J Hered 92:295–300

Kunzel G, Korzun L, Meister A (2000) Cytologically integrated physicalrestriction fragment length polymorphism maps for the barleygenome based on translocation breakpoints. Genetics 154:397–412

Lai CW, Yu Q, Hou S, Skelton RL, Jones MR, Lewis KL, Murray J,Eustice M, Guan P, Agbayani R, Moore PH, Ming R, PrestingGG (2006) Analysis of papaya BAC end sequences reveals firstinsights into the organization of a fruit tree genome. Mol GenetGenomics 276:1–12

Lengerova M, Kejnovsky E, Hobza R, Macas J, Grant SR, Vyskot B(2004) Multicolor FISH mapping of the dioecious model plant,Silene latifolia. Theor Appl Genet 108:1193–1199

Lim KY, Matyasek R, Lichtenstein CP, Leitch AR (2000) Molecularcytogenetic analyses and phylogenetic studies in the Nicotianasection Tomentosae. Chromosoma 109:245–258

Lysak MA, Fransz PF, Ali HB, Schubert I (2001) Chromosomepainting in Arabidopsis thaliana. Plant J 28:689–697

Ma H, Moore PH, Liu Z, Kim MS, Yu Q, Fitch MM, Sekioka T,Paterson AH, Ming R (2004) High-density linkage mappingrevealed suppression of recombination at the sex determinationlocus in papaya. Genetics 166:419–436

McClintock B (1931) The order of the genes C, Sh and Wx in ZeaMays with reference to a cytologically known point in thechromosome. Proc Natl Acad Sci USA 17:485–491

McClintock B, Hill HE (1931) The cytological identification of thechromosome associated with the R-G linkage group in ZEAMAYS. Genetics 16:175–190

Milla R, Gustafson JP (2001) Genetic and physical characterization ofchromosome 4DL in wheat. Genome 44:883–892

180 Tropical Plant Biol. (2010) 3:171–181

Ming R, Moore PH, Zee FT, Abbey CA, Ma H, Paterson AH (2001)Construction and characterization of a papaya BAC library as afoundation for molecular dissection of a tree-fruit genome. TheorAppl Genet 102:892–899

Ming R et al (2008) The draft genome of the transgenic tropical fruittree papaya (Carica papaya Linnaeus). Nature 452:991–996

Nagarajan N, Navajas-Perez R, Nihai P, Alam M, Ming R, PatersonAH, Salzberg SL (2008) Genome-wide analysis of repetitiveelements in papaya. Tropical Plant Biol 1:191–202

Paterson AH et al (2009) The Sorghum bicolor genome and thediversification of grasses. Nature 457:551–556

Pedrosa A, Vallejos CE, Bachmair A, Schweizer D (2003) Integrationof common bean (Phaseolus vulgaris L.) linkage and chromo-somal maps. Theor Appl Genet 106:205–212

Peters SA, Datema E, Szinay D, van Staveren MJ, Schijlen EG, vanHaarst JC, Hesselink T, Abma-Henkens MH, Bai Y, de Jong H,Stiekema WJ, Klein Lankhorst RM, van Ham RC (2009)Solanum lycopersicum cv. Heinz 1706 chromosome 6: distribu-tion and abundance of genes and retrotransposable elements.Plant J 58:857–869

Portis E, Mauromicale G, Mauro R, Acquadro A, Scaglione D, LanteriS (2009) Construction of a reference molecular linkage map ofglobe artichoke (Cynara cardunculus var. scolymus). Theor ApplGenet 120:59–70

Ren Y, Zhang Z, Liu J, Staub JE, Han Y, Cheng Z, Li X, Lu J, MiaoH, Kang H, Xie B, Gu X, Wang X, Du Y, Jin W, Huang S (2009)An integrated genetic and cytogenetic map of the cucumbergenome. PLoS ONE 4:e5795

Robledo G, Lavia GI, Seijo G (2009) Species relations among wildArachis species with the A genome as revealed by FISH mappingof rDNA loci and heterochromatin detection. Theor Appl Genet118:1295–1307

Rosato M, Castro M, Rossello JA (2008) Relationships of the woodyMedicago species (section Dendrotelis) assessed by molecularcytogenetic analyses. Ann Bot 102:15–22

Rozen S, Skaletsky H (2000) Primer3 on the WWW for general usersand for biologist programmers. In: Krawetz S, Misener S (eds)Bioinformatics methods and protocols: methods in molecularbiology. Humana Press, Totowa, pp 365–386

Saintenac C, Falque M, Martin OC, Paux E, Feuillet C, Sourdille P(2009) Detailed recombination studies along chromosome 3Bprovide new insights on crossover distribution in wheat (Triticumaestivum L.). Genetics 181:393–403

Schubert I, Fransz PF, Fuchs J, de Jong JH (2001) Chromosomepainting in plants. Methods Cell Sci 23:57–69

Shishido R, Sano Y, Fukui K (2000) Ribosomal DNAs: an exceptionto the conservation of gene order in rice genomes. Mol GenetGenomics 263:586–591

Storey WB (1953) Genetics of papaya. J Heredity 44:70–78Taketa S, Harrison GE, Heslop-Harrison JS (1999) Comparative

physical mapping of the 5S and 18S-25S rDNA in nine wildHordeum species and cytotypes. Theor Appl Genet 98:1–9

Tang X, Szinay D, Lang C, RamannaMS, van der Vossen EA, Datema E,Lankhorst RK, de Boer J, Peters SA, BachemC, StiekemaW, VisserRG, de Jong H, Bai Y (2008) Cross-species bacterial artificialchromosome-fluorescence in situ hybridization painting of thetomato and potato chromosome 6 reveals undescribed chromosomalrearrangements. Genetics 180:1319–1328

Tang X, de Boer JM, van Eck HJ, Bachem C, Visser RG, de Jong H(2009) Assignment of genetic linkage maps to diploid Solanumtuberosum pachytene chromosomes by BAC-FISH technology.Chromosome Res 17:899-915

Walling JG, Shoemaker R, Young N, Mudge J, Jackson S (2006)Chromosome-level homeology in paleopolyploid soybean (Gly-cine max) revealed through integration of genetic and chromo-some maps. Genetics 172:1893–1900

Wang Y, TangX, Cheng Z,Mueller L, Giovannoni J, Tanksley SD (2006)Euchromatin and pericentromeric heterochromatin: comparativecomposition in the tomato genome. Genetics 172:2529–2540

Wang K, Guo W, Zhang T (2007) Development of one set ofchromosome-specific microsatellite-containing BACs and theirphysical mapping in Gossypium hirsutum L. Theor Appl Genet115:675–682

Wang K, Guan B, Guo W, Zhou B, Hu Y, Zhu Y, Zhang T (2008)Completely distinguishing individual A-genome chromosomesand their karyotyping analysis by multiple bacterial artificialchromosome—fluorescence in situ hybridization. Genetics178:1117–1122

Watson JD, Crick FH (1953) Molecular structure of nucleic acids; astructure for deoxyribose nucleic acid. Nature 171:737–738

Yu Q, Tong E, Skelton RL, Bowers JE, Jones MR, Murray JE, Hou S,Guan P, Acob RA, Luo MC, Moore PH, Alam M, Paterson AH,Ming R (2009) A physical map of the papaya genomewith integratedgenetic map and genome sequence. BMC Genomics 10:371

Zhang D, Yang Q, Bao W, Zhang Y, Han B, Xue Y, Cheng Z (2005)Molecular cytogenetic characterization of the Antirrhinum majusgenome. Genetics 169:325–335

Zhang W, Wang X, Yu Q, Ming R, Jiang J (2008) DNA methylationand heterochromatinization in the male-specific region of theprimitive Y chromosome of papaya. Genome Res 18:1938–1943

Tropical Plant Biol. (2010) 3:171–181 181